Bacterial pathogens rose to the challenge of antimicrobial agents by developing drug resistance through two types of mechanisms (i) an adaptive mechanism in which the bacterial pathogen "left to it's own devices" undergoes genetic changes (point mutations, regulatory changes, rearrangements etc.) under the selective pressure of the antibiotic. One may refer to this process as evolution of drug resistance. The second type of mechanism involves acquisition of resistance determinants from heterologous sources and incorporation of these into the regulatory circuitry of the particular pathogen. This research proposal will dissect genetic, biochemical, regulatory and cell biological aspects of resistance mechanisms in S. aureus concentrating on two of the most important antimicrobial agents used against this bacteria: b-lactam antibiotics and vancomycin. Both beta-lactam antibiotics and vancomycin target the bacterial cell wall. Therefore, elucidation of the mechanism of drug resistance should also provide important insights into the mechanism and organization of cell wall biosynthesis and assembly. Project A: Mechanism of VISA type resistance in S. aureus. An evolutionary type of vancomycin resistance will be studied in a series of isogenic MRSA isolates that developed vancomycin resistance during chemotherapy of a patient. Complete DNA sequencing of the susceptible parental and the vancomycin resistant mutant isolate; computational analysis of sequence differences; characterization of gene expression profiles by DNA microarrays, localization of cell wall synthetic sites and genetic crosses should lead to a better understanding of the mechanism of resistance and allow reconstruction of stages in the evolutionary pathway leading to the vancomycin resistant phenotype. Project B: Expression of the van A gene complex in S. aureus. The mechanism of acquired vancomycin resistance will be examined by genetic, biochemical and RNA profiling experiments in a VRSA strain which carries both the enterococcal vanA gene complex and the b-lactam resistance gene mecA. Project C: Integration and functioning of the acquired mecA gene in the S. aureus host will be studied following up the finding of functional cooperation between the resistance protein PBP2A and the native PBP2 of S. aureus: Direct interaction between the two proteins; their co-localization at subcellular sites of wall biosynthesis and the possible co-regulation of expression of pbpB and the mecA determinant will be tested. Project D: From resistance gene to resistant phenotype. An evolutionary type of process appears to be critical for the "grafting" of the b-lactam resistance gene mecA into the S. aureus cells and for the capacity of the bacteria to express high level resistance - as manifested in the unique heterogeneous phenotype of many MRSA clinical isolates. Attempts will be made to identify the number, nature and mode of action of domestic genes which are recruited in this process - using genetic crosses and microarray profiling combined with data analysis by modern biomathematical approaches.